Protein kinases participate in the signaling events which control the activation, growth, differentiation, survival and migration of cells in response to extracellular mediators or stimuli including growth factors, cytokines or chemokines. In general, these kinases are classified in two groups, those that preferentially phosphorylate tyrosine residues and those that preferentially phosphorylate serine and/or threonine residues. The tyrosine kinases include membrane-spanning growth factor receptors, for example the epidermal growth factor receptor (EGFR) and cytosolic non-receptor kinases including Src family kinases, the Syk family kinases and the Tec family kinases.
Inappropriately high protein kinase activity is involved in many diseases including cancer, metabolic diseases, immunological diseases and inflammatory disorders. This can be caused either directly or indirectly by the failure of control mechanisms due to mutation, overexpression or inappropriate activation of the enzyme.
Protein tyrosine kinases—both receptor tyrosine kinases and non-receptor kinases—are essential for the activation and proliferation of cells of the immune system. Among the earliest detectable events upon the immunoreceptor activation in mast cells, T cells and B cells is the stimulation of non-receptor tyrosine kinases.
Phosphoinositide 3-kinases (PI3Ks) were early on identified as lipid kinases associated with viral oncogens [Whitman et al., Nature 315:239-242 (1985); Sugimoto et al., Proc. Natl. Acad. Sci. 81:2117-2121 (1984); Macara et al., Proc. Natl. Acad. Sci. 81:2728-2732 (1984)], and for the last 20 years, the connection between cancer and PI3K has been further substantiated [Cully et al., Nat. Rev., Cancer 6:184-192 (2006); Wymann et al., Curr. Opin. Cell Biol. 17:141-149 (2005); Vivanco et al., Nat. Rev., Cancer 2:489-501 (2002)]. PI3Ks have since been recognized to modulate a wide range of cellular activities, and to be central to the growth and metabolic control. Genetically modified mice targeting the PI3K pathway, and the elucidation of human hereditary disease like Cowden's syndrome, tuberous sclerosis, ataxia telangiectasia, X-linked myotubular myopathy and Charcot-Marie-Tooth neuropathy, have provided further insight in the cellular and systemic role of phosphoinositide signaling. Deregulation of phosphoinositide levels, and in particular the product of class I PI3Ks, PtdIns (3,4,5)P3, is involved in the pathogenesis of cancer, chronic inflammation, allergy, metabolic disease, diabetes and cardiovascular problems.
PI3Ks are a family of enzymes, which phosphorylate the 3′-OH position of the inositol ring of phosphoinositides. They have been divided into three classes on the basis of structural features and in vitro lipid substrate specificity [(Marone et al, Biochimica et Biophysica Acta 1784:159-185 (2008)]. Class I PI3Ks form heterodimers, which consist of one of the four closely related ˜110 kDa catalytic subunits, and an associated regulatory subunit belonging to two distinct families. In vitro they are capable to convert PtdIns to PtdIns-3-P, PtdIns-4-P to PtdIns(3,4)P2, and PtdIns(4,5)P2 to PtdIns(3,4,5)P3, but the in vivo substrate is PtdIns(4,5)P2 [Cantley et al., Science 296:1655-1657 (2002)]. Class I PI3Ks are activated by a large variety of cell-surface receptors, comprising growth factor receptors as well as G protein-coupled receptors.
Class II PI3Ks are capable to phosphorylate PtdIns and PtdIns-4-P in vitro, but their relevant in vivo substrates are still under investigation. This class of large (170-200 kDa) enzymes has three members, all characterized by a C-terminal C2 homology domain. No adaptor molecules for class II PI3Ks have been identified so far. Class III PI3Ks are solely able to phosphorylate PtdIns, and thus generate only PtdIns-3-P. The single member of this class is Vps34, of which the S. cerevisiae Vps34p (vacuolar protein sorting mutant 34 protein) is the prototype, and has been shown to play an essential role in trafficking of newly synthesized proteins from the Golgi to the yeast vacuole, an organelle equivalent to lysosomes in mammals [Schu et al., Science 260:88-91 (1993)].
Phosphoinositide 4-kinases (PI4Ks) phosphorylate the 4′-OH position of the inositol ring of PtdIns, and thereby generate PtdIns-4-P. This lipid can then be further phosphorylated by PtdIns-4-P 5-kinases to generate PtdIns (4,5)P2, which is the main source for phospholipase C and PI3K signaling at the plasma membrane. Four PI4Ks isoforms are known: PI4KIIα and β, and PI4KIIIα and β. The PI4KIIIs are most closely related to PI3Ks.
The class of PI3K-related proteins, referred to as class IV PI3Ks, consists of high molecular weight enzymes with a catalytic core similar to PI3Ks and PI4Ks and include the target of rapamycin (mTOR, also known as FRAP), DNA-dependent protein kinase (DNA-PKcs), the ataxia telangiectasia mutated gene product (ATM), ataxia telangiectasiarelated (ATR), SMG-1 and transformation/transcription domain-associated protein (TRRAP). The first five members are active protein serine-threonine kinases that are involved in cell growth control and genome/transcriptome surveillance [(Marone et al., Biochimica et Biophysica Acta 1784:159-185 (2008)]. DNA-PKcs, ATM, ATR and SMG-1 are involved in DNA-damage responses. The only active kinase not involved in DNA-damage is mTOR, which is regulated by growth factors and nutrient availability, and coordinates protein synthesis, cell growth and proliferation. Target of rapamycin (mTOR) complexes 1 and integrate growth factor signaling (via PI3K/PKB and the Ras/MAPK cascade), energy status (LKB1 and AMPK) and nutrient detection. TOR is positively regulated by PKB/Akt, which phosphorylates the negative regulator TSC2 in the tuberous sclerosis complex (TSC), resulting in activation of the GTPase Rheb and mTOR [(Marone et al., Biochimica et Biophysica Acta 1784:159-185 (2008)]. In parallel, mTOR stimulates translation of ribosomal proteins and therefore ribosome biogenesis via the activation of p70S6K [Wullschleger et al., Cell 124:471 (2006)]. Rapamycin, and its derivatives RAD001 and CCI-779, bind to FKBP12, and the complex blocks mTOR complex 1 (mTORC1) activity very selectively. Various clinical trials were initiated using Rapamycin and derivatives, mostly in patients with tumors displaying elevated PI3K signaling and hyperactive mTOR. Promising results were obtained in mantle cell lymphoma, endometrial cancer and renal cell carcinoma [Guertin et al., Cancer Cell 12:9 (2007)]. Rapamycin and its derivatives possess anti-angiogenic activity because they counteract VEGF action [Guba et al., Nat. Med. 8:128 (2002)]. This opens avenues for combinatorial treatments with conventional chemotherapy [Beuvink et al., Cell 120:747 (2005)].
The PI3K pathway is a key signaling transduction cascade controlling the regulation of cell growth, proliferation, survival as well as cell migration. PI3Ks are activated by a wide variety of different stimuli including growth factors, inflammatory mediators, hormones, neurotransmitters, and immunoglobulins and antigens [Wymann et al., Trends Pharmacol. Sci. 24:366-376 (2003)]. The class IA PI3K isoforms PI3Kα, β and δ, are all bound to one of the p85/p55/p50 regulatory subunits, which all harbor two SH2 domains that bind with high affinity to phosphorylated Tyr-X-X-Met motifs. These motifs are present in activated growth factor receptors, their substrates and numerous adaptor proteins. As described above, activation of the PI3K/PKB signaling cascade has a positive effect on cell growth, survival and proliferation. Constitutive up-regulation of PI3K signaling can have a deleterious effect on cells leading to uncontrolled proliferation, enhanced migration and adhesion-independent growth. These events favor not only the formation of malignant tumors, but also the development of inflammatory and autoimmune disease.
The PI3 kinase/Akt/PTEN pathway is an attractive target for cancer drug development since such agents would be expected to inhibit proliferation, reverse the repression of apoptosis and surmount resistance to cytotoxic agente in cancer cells. PI3 kinase inhibitors have been reported [see notably Marone et al., Biochimica et Biophysica Acta 1784:159-185 (2008); Yaguchi et al. (2006) Jour. Of the Nat. Cancer Inst. 98(8):545-556; U.S. Pat. No. 7,173,029; U.S. Pat. No. 7,037,915; U.S. Pat. No. 6,608,056; U.S. Pat. No. 6,608,053; U.S. Pat. No. 6,838,457; U.S. Pat. No. 6,770,641; U.S. Pat. No. 6,653,320; U.S. Pat. No. 6,403,588; U.S. Pat. No. 6,703,414; WO9715658; WO2006046031; WO2006046035; WO2006046040; WO2007042806; WO2007042810; WO2004017950; US2004092561; WO2004007491; WO2004006916; WO2003037886; US2003149074; WO2003035618; WO2003034997; WO2007084786; WO2007095588; WO2008098058; US2003158212; EP1417976; US2004053946; JP2001247477; JP08175990; JP08176070].
1,3,5-triazine and pyrimidine derivatives as pharmaceuticals have been made with respect to antitumor, anti-inflammatory, analgesic and antispasmodic activities. Especially, hexamethylmelamine or altretamin (HMM or N2,N2,N4,N4,N6,N6-hexamethyl-1,3,5-triazine-2,4,6-triamine) is well-known, which has been developed as analogue of antitumor agent triethylenemelamine (TEM); HMM acts as a prodrug of hydroxymethylpentamethylmelamine (HMPMM: metabolically active type of HMM) [Johnson et al., Cancer, 42:2157-2161 (1978)]. HMM has been marketed in Europe under the indications for the treatment of ovarian and small cell lung cancers.
Certain triazine compounds are known to have PI3 kinase inhibitor activity and inhibit the growth of cancer cells [WO02088112 (EP1389617), “HETEROCYCLIC COMPOUNDS AND ANTITUMOR AGENT CONTAINING THE SAME AS ACTIVE INGREDIENT”, Kawashima et al., Filing date: 26 Apr. 2002; WO05095389 (EP1741714), “HETEROCYCLIC COMPOUND AND ANTI-MALIGNANT-TUMOR AGENT CONTAINING THE SAME AS ACTIVE INGREDIENT”, Kawashima et al., Filing date: 30 Mar. 2005; WO06095906 (EP1864665), “IMMUNOSUPPRESSIVE AGENT AND ANTI-TUMOR AGENT COMPRISING HETEROCYCLIC COMPOUND AS ACTIVE INGREDIENTS”, Haruta et al., Filing date: 11 Mar. 2005; WO09905138 (EP1020462), HETEROCYCLIC COMPOUNDS AND ANTITUMOR AGENT CONTAINING THE SAME AS ACTIVE INGREDIENT, Kawashima et al., Filing date: 24 Jul. 1998;]. The triazine compound ZSTK474, developed in research laboratories of Zenyaku Kogyo is the first orally administered triazine compound highly active against PI3Ks that displayed potent antitumor activity against human cancer xenografts in mice, without evidence of critical toxicity [Yaguchi et al., Journal of the National Cancer Institute, 98:545-556, (2006)]. ZSTK474 is an ATP-competitive inhibitor of class I phosphatidylinositol 3-kinase isoforms [Kong et al., Cancer Sci, 98:1638-1642 (2007)].
Certain pyrimidine compounds are known to have p110 alpha binding, PI3 kinase inhibitor activity and inhibit the growth of cancer cells [IP of AstraZeneca: WO07066103, WO07080382, WO08023159, WO08023180, WO08032027, WO08032033, WO08032036, WO08032041, WO08032072, WO08032077, WO08032086, WO08032089, WO08032091; IP of Genentech/Piramed/Roche: US2007009880, WO07127183, WO08073785, WO07042810, WO07122410, WO07127175, WO07129161, WO08070740, WO2006046031, WO2006046040, WO2007042806, WO2007122410; IP of Novartis: WO07084786, WO08098058].
In order to expand antitumor spectrum of and increase antitumor activities of such compounds, active against PI3Ks and/or mTOR, the inventors carried out intensive studies on triazine-, pyrimidine- and pyridine-based derivatives. They thus prepared new heterocyclic compounds represented by the formula (I) and formulas (Ia) to (Ii) which exhibit strong biological activity against lipid kinases.
In comparison with the PI3K inhibitors disclosed by Zenyaku Kogyo [WO02088112 (EP1389617), WO2005095389 (EP 1741714), WO2006095906 (EP1864665), WO09905138 (EP1020462)], AstraZeneca (WO07066103, WO07080382, WO08023159, WO08023180, WO08032027, WO08032033, WO08032036, WO08032041, WO08032072, WO08032077, WO08032086, WO08032089, WO08032091), Piramed/Genentech (US2007009880, WO07127183, WO08073785, WO07042810, WO07122410, WO07127175, WO08070740, WO2006046031, WO2006046040, WO2007122410), Yamanouchi/Piramed (WO01083456) and Novartis (WO07084786, WO08098058), the inhibitors of the invention differ in the insertion of a N atom in the basic heterocyclic ring that makes better biological activity to the target enzyme, and/or in the insertion of a novel molecular fragment making the whole molecule more active or more selective to the appropriate enzyme.